Isolation, Purification And Structural Characterization Of The Polysaccharide LBLP5-A From Lycium Barbarum Leaves

Lycium barbarum L., a solanaceous deciduous shrub plant, is a kind of famous traditional medicine and food plants. Its fruits, leaves and root all are medicine in China. In the medicinal monographs "ben cao gang mu" and "li dai ben cao", L. barbarum leaves are called as "tian jing cao" and demonstrated to have a variety biological activities on enhancing immunity, clearing heat, slaking thirsty, producing saliva, antioxidant and improving eyesight. Polysaccharide may be important active constituents of Lycium barbarum leaves.In this study, LBLP5-A were extracted and isolated from Lycium barbarum leaves, physical and chemical properties, structural characterization, In vitro antioxidant activity of LBLP5-A were researched, the conclusions were as below:1. Extraction, isolation and purification, physical and chemical properties of LBLP5-ACrude polysaccharide (LBLP) was extracted by hot water method and alcohol precipitation. The LBLP was separated and further purified by DEAE-cellulose chromatography and Sephadex G-100 column, LBLP5-A was obtained. The physical and chemical properties of LBLP5-A was characterized by UV、HPLC、GC、IR. The results were that LBLP5-A was homogeneity, and the average molecular weight (MW) of LBLP5-A was estimated to be 11.33×104 Da; the LBLP5-A was made up of rhamnose, arabinose, and galactose in the molar ratio of 0.5:1.9:1.0; the total sugar content was 93.71%, and the protein content was 4.6%.2. Studies on structural characterization of LBLP5-AThe existence of O-type carbohydrate-peptide linkage in LBLP5-A was demonstrated by β-elimination reaction. The fractions LBLP5-A-OL1, LBLP5-A-OL2, LBLP5-A-OL3, LBLP5-A-OL4 were obtained by releasing glycan from LBLP5-A. Detailed structure patterns of LBLP5-A-OL1 was characterized based on monosaccharide composition analysis, methylation analysis, ESI-MS and partial hydrolysis. The results revealed that LBLP5-A-OL1 have a backbone of (1�'3)-linked Gal partially substituted at C-6 position with -6)Gal(1-, Gal(1- and -4)Gal(1-. The branches were composed of Ara(1-,-3)Ara(1-,-5)Ara(1- and -2, 4)Rha(1-. LBLP5-A-OL3 and LBLP5-A-OL4 were characterized by ESI-MS. LBLP5-A-OL3 and LBLP5-A-OL4 were both composed of a series of oligosaccharide that share the general structure pattern of Aran1�'Galn2�'Rhan3 (nl=0-6, n2=0-8, n3=0-1).3. Anti-oxidant activity of LBLP5-AThe LBLP5-A illustrated excellent anti-oxidant activity as evaluated concerning reducing power and the scavenging abilities on superoxide, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals in vitro. At the concentration of 2.0 mg/mL, the scavenging activities of DPPH radical with LBLP5-A was 54.5% and weaker than VC. The scavenging of superoxide radicals of LBLP5-A was higher than that of Vc at the concentration (>0.6 mg/mL). VC and LBLP5-A were 69.23% and 83.68% at 2.0 mg/mL. The scavenging of hydroxyl radical and reducing power with LBLP5-A was higher than that of Vc at a higher concentration (>2.5 mg/mL).